Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-10-25
2004-07-06
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S029000
Reexamination Certificate
active
06759201
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the Daedalos nucleic acids, Daedalos polypeptides, and other related molecules and methods of making and using the same.
BACKGROUND OF THE INVENTION
The maintenance of tissues that require regeneration during the life of an organism is often achieved by the asymmetric division of a less differentiated stem cell to regenerate itself as well as give rise to a daughter cell that can then differentiate to repopulate the organ. The best characterized stem cells in the adult animal are those that regenerate the hematopoietic system. The production or proliferation of the hematopoietic stem cells (HSCs), and the subsequent expansion of progenitors with progressively restricted developmental potential derived from them, is regulated in part by members of the Ikaros gene family (Georgopoulos et al. (1997) Annu. Rev. Immunol. 15:155). Ikaros, Aiolos and Helios comprise the previously identified members of the Ikaros gene family. They encode conserved zinc finger DNA binding proteins which are expressed at varying levels in cells progressing through the hematopoietic lineages (Kelley et al. (1998) Curr. Biol, 8:508). Mutations in Ikaros cause defects in the hematopoietic stem cell as well as in later stages of lymphoid differentiation (Georgopoulos et al. (1994) Cell 79:143), while Aiolos mutations cause defects which are restricted to the lymphoid lineages, particularly in the sub-lineage that gives rise to B cells (Wang et al. (1998) Immunity 9:543).
Co-localization studies on the Ikaros family proteins suggest that these proteins bind to lineage specific genes in lymphoid cells and may serve to mediate rapid transitions between subsequently heritable repressed and active states in response to extrinsic signals. In support of this model, both Ikaros and Aiolos assemble into at least two distinct chromatin remodeling complexes (Kim et al. (1999) Immunity 10:345). One of these includes Mi-2 and histone deacetylase (HDAC) and can assemble chromatin in a closed conformation while the other includes members of a SWI/SNF complex associated with chromatin opening. Ikaros family proteins also regulate proliferative responses in maturing T cells, possibly by regulating access of the replication machinery to DNA (Avitahl et al. (1999) Immunity 10:333). These observations led to the general model that changes in the combinatorial expression of Ikaros family members during progression through the lymphoid lineage regulate the gene expression changes associated with successive steps in lymphoid development (Kelley et al. (1998) Curr. Biol. 8:508-515).
SUMMARY OF THE INVENTION
The invention is based, in part, on the discovery that Daedalos, a member of the Ikaros family of proteins, is differentially expressed at various stages of neural cell maturation. It was found that forced expression of Daedalos affected neural cell differentiation.
In general, the invention features a method of characterizing or detecting a cell, e.g., a neural cell, e.g., a neural progenitor cell, e.g., a neural progenitor cell in a cell sample. The method includes: providing a cell; and detecting the absence or presence of expression of Daedalos in the cell, wherein expression of Daedalos is indicative of a neural progenitor cell, to thereby characterize or detect a cell, e.g., a neural progenitor cell. The method can further include isolating or purifying the cell.
In one embodiment, the cell sample includes non-neural cells. The non-neural cells can be of any cell type. Non-neural cells can be included in the cell sample by extracting the cell sample from tissue of a subject, wherein the extraction results in a heterogeneous population of cells. Examples of non-neural cells that can be included in the cell sample are fibroblasts, epithelial cells, and hematopoietic cells. The method can be performed in vitro or in vivo.
In one embodiment, the absence or presence of a Daedalos mRNA is detected in the cell. Various techniques known to one of skill in the art can be used to detect a Daedalos mRNA. For example, a Daedalos mRNA can be detected by using a nucleic acid probe that hybridizes to a Daedalos mRNA. A detectable label, e.g., a radioactive or fluorescent label, can optionally be attached to the nucleic acid probe in this detection method. In another example, a Daedalos mRNA can be detected by PCR. Detection by PCR can include a further step of hybridization of a nucleic acid probe, e.g., a labeled nucleic acid probe, to the PCR product.
In one embodiment, the absence or presence of a Daedalos protein is detected. A Daedalos protein can be detected by various techniques known to one of skill in the art. For example, an antibody can be used that binds to a Daedalos protein. A detectable label, e.g., a radioactive or fluorescent label, can be attached to the antibody that binds to a Daedalos protein. Other known methods of protein detection include Western blot immunoassay, immunohistology, fluorescence activated cell sorting (FACS), radioimmunoassay (RIA), fluorescent immunoassay, enzyme linked immunosorbent assay (ELISA), or an immunoassay that uses a solid support, e.g., latex beads.
Expression of Daedalos can be used as a marker to characterize, detect, separate or purify cells.
In another embodiment, the method further includes separating the neural progenitor cell from at least one non-neural progenitor cell present in the cell sample. According to this method, the neural progenitor cell can be separated from other cells based upon expression of Daedalos detected in the neural progenitor cell.
In another embodiment, Daedalos expression is detected by providing a cell in which a Daedalos control region is functionally coupled to a nucleic acid which encodes a protein other than Daedalos, e.g., a reporter molecule, e.g., lacZ or a fluorescent product, e.g., green fluorescent protein. Expression can be used to follow development in a system, e.g., in a mouse, nematode, fish (e.g., a zebrafish), e.g., in a transgenic animal, e.g., a transgenic mouse, nematode or zebrafish.
In another aspect, the invention features a method of separating a neural progenitor cell from a cell population. The method includes: providing a cell population, e.g., two or more cells, containing a neural progenitor cell and a non-neural progenitor cell; evaluating expression of Daedalos in the neural progenitor cell and in the non-neural progenitor cell; and separating the neural progenitor cell from the non-neural progenitor cell based upon their expression of Daedalos. The cell population can be derived from neural tissue, e.g., glial cells. The cell population can contain neural and non-neural cells.
In one embodiment, the neural progenitor cell has a higher level of expression of Daedalos as compared to the non-neural progenitor cell.
In one embodiment, levels of Daedalos mRNA produced in the neural progenitor cell and in the non-neural progenitor cell are evaluated. Levels of Daedalos mRNA can be evaluated by various techniques known by one of skill in the art. In one example, levels of Daedalos mRNA are evaluated by a nucleic acid probe that hybridizes to the Daedalos mRNA. The nucleic acid probe can optionally include a detectable label attached to the nucleic acid probe. In another example, Daedalos mRNA is detected by PCR, as described herein. Additionally, Daedalos expression can be evaluated by detecting the level of Daedalos protein expression by the neural progenitor cell and the non-neural progenitor cell. In one example, the Daedalos protein is detected by an antibody that binds to the Daedalos protein. The antibody can optionally include a detectable label attached thereto. Other known methods of protein detection include Western blot immunoassay, immunohistology, fluorescence activated cell sorting (FACS), radioimmunoassay (RIA), fluorescent immunoassay, enzyme linked immunosorbent assay (ELISA), or an immunoassay that uses a solid support, e.g., latex beads.
In another aspect, the invention features a method of identifying the stage of neurogenesis of a cell. The method includes:
Fish & Richardson P.C.
McKelvey Terry
The General Hospital Corporation
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